Adrenomedullin and its receptors are expressed in mouse pancreatic β-cells and suppresses insulin synthesis and secretion

Gestational diabetes mellitus (GDM) is associated with defective pancreatic β-cell adaptation in pregnancy, but the underlying mechanism remains obscure. Our previous studies demonstrated that GDM women display increased plasma adrenomedullin (ADM) levels, and non-obese GDM mice show decreased serum concentrations of insulin and the number of β-cells in pancreas islets. The aims of this study is to examine if ADM and its receptors are expressed in female mouse pancreas, and if so, whether insulin secretion is regulated by ADM in mouse β-cell line, NIT-1 cells and isolated mouse pancreatic islets. Present study shows that ADM and its receptor components CRLR, RAMPs are present in mouse pancreatic islets and co-localized with insulin. The expressions of ADM, CRLR and RAMP2 in islets from pregnant mice are reduced compared to that of non-pregnant mice. NIT-1-β cells express ADM and its receptor mRNA, and glucose dose-dependently stimulates expressions. Furthermore, ADM inhibits NIT-1-β cell growth, and this inhibition is reversed by ADM antagonist, ADM22-52. The glucose-induced insulin secretion was suppressed by ADM in NIT-1-β cells and isolated pancreatic islets from pregnant mice. These inhibitory effects are accompanied by upregulation of endoplasmic reticulum (ER) stress biomarker genes in NIT-1-β cells. This study unveils that reduced ADM and its receptors may play a role in β-cell adaptation during pregnancy, while increased plasma ADM in GDM may contribute to the β-cells dysfunction, and blockade of ADM may reverse β-cell insulin production.


Introduction
During normal pregnancy, marked insulin resistance is accompanied by increased maternal insulin secretion [1]. This increased insulin resistance may contribute to facilitate the transportation of glucose and other nutrients toward the fetus. Maternal islets adapt to this increased demand mainly through increased β-cell proliferation and enhanced insulin secretion per βcell [2]. Failure of these compensatory mechanisms may result in the development of glucose

Glucose Stimulated Insulin Secretion (GSIS) assays
Pancreatic islets were isolated from day 13.5 pregnant mice (n = 4) as previously described [20] and used routinely in our labs. Briefly, pancreas was infused with collagenase, removed and incubated in collagenase solution at 37˚C. Once digestion was complete, samples were washed and ficoll gradient was applied to the isolated tissue. Cell pellets were resuspended in RPMI 1640. Islets were then picked, graded by size, and placed into culture overnight without or with ADM (10 -5 M). The following day islets were picked and placed in tubes with low (1.8 mM) or high glucose (16.8 mM) and with and without ADM. Media was collected following 30 min of culture to measure secreted insulin. Secreted and total insulin were measured by ELISA (Millipore) as previously described [16,21,22]. Percent secreted insulin was calculated and compared between groups to determine the effects of increased ADM on GSIS in islets from pregnant mice.

Cell proliferation assay
The cell proliferation assay was performed by using Cell proliferation Kit I (MTT) (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacture's instruction. Briefly, NIT-1 cells were seeded in wells of 96-well plate containing various amounts of ADM (American Peptide Co., Inc. Sunnyvale, CA, USA) with or without ADM22-52 (American Peptide Co., Inc. Sunnyvale, CA, USA). After 72 hours preincubation, 10 μl of MTT labeling reagent was added and incubated for another 4 hours, followed by the treatment with solubilization solution for overnight. The absorbance at A550 were read and recorded by using Spectrophotometer CLARIO STAR (BMG Labtech, Inc., Cary, NC, USA).

Insulin analysis
Insulin concentrations in NIT-1 cell culture medium were assessed using a Mouse insulin ELISA kit (Thermo Scientific, Frederick, MD, USA) according to manufacturer's instructions. Briefly, NIT-1 cells were seeded in 96-well plates. After 3 days culture, the cells were treated with increasing doses of glucose (Sigma-Aldrich, St. Louis, MO, USA) in the presence or absence of ADM (10 −9 M, American Peptide Co., Inc), and culture medium was collected 30-min and 24 hours after treatments. For insulin measurement, 100 μL of standards and culture medium were added into appropriate wells and incubate for 2 hours at room temperature, followed by incubation with biotinylated antibody, streptavidin-HRP solution and TMB substrate. The absorbance at A450 were read and recorded by using Spectrophotometer CLARIO STAR (BMG Labtech, Inc., Cary, NC, USA). The intra-assay coefficients of variation were <10% and intra-assay CVs were <12%.

Statistics
All data were presented as mean ± SEM. Data were calculated and analyzed by GraphPad Prism (La Jolla, CA, USA). For comparison between 2 groups unpaired 2-tailed Student's t test was used. For comparisons between 3 or more groups, a one-way ANOVA was used followed

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https://doi.org/10.1371/journal.pone.0265890.t001 by a Bonferroni post hoc test was used for comparisons between groups. For analysis of mRNA expressions data, two-way repeated measures ANOVA was used, followed by Bonferroni post hoc analysis where appropriate. Statistical significance was defined as p<0.05.

ADM and its receptor components are present in mouse pancreatic islets and co-localized with insulin
Triple immunofluorescence was applied to investigate the co-localization of the ADM and its receptor components with classical pancreatic hormones, glucagon and insulin, thereby identify the cell type in which these epitopes are expressed. In all cases, co-localization with insulin was observed for ADM, CRLR, RAMP2, and RAMP3 in the islets of pancreatic tissues from both non-pregnant and pregnant mice (Figs 1 and 2), whereas their immunoreactivity was rarely observed glucagon producing α cells, suggesting that a new endocrine function in the mouse pancreas, insulin-producing β-cells might be synthesizing and secreting ADM. Furthermore, the intensity of immunostaining for target proteins showed that no significant difference in the expressions of ADM and its receptor components was noted between animals on day 13.5 and day 17.5 of pregnancy (Figs 1 and 2), however, the expression for ADM, CRLR and RAMP2, but not RAMP3, are significantly reduced (p<0.05) in islets from pregnant mice when compared to that of non-pregnant mice, suggesting that alteration of ADM system in mouse pancreas may play a role in pregnancy-related β-cell adaptation.

ADM blocks glucose stimulated insulin secretion in pancreatic islets isolated from pregnant mice
Next, we assessed the effect of ADM on glucose stimulated insulin secretion using isolated pancreatic islets from day 13.5 pregnant mice. Islets cultured with ADM had inhibited GSIS while those cultured in saline had normal GSIS (Fig 3). These results indicate that increases in ADM levels can have negative effects on insulin secretion. To further evaluate the mechanisms by which ADM effects insulin secretion we transitioned to using the mouse β-cell line, NIT-1.

NIT-1 β-cells express mRNA for ADM and its receptor components, and glucose stimulates the expressions for CRLR, RAMP2, and RAMP3
To determine the impact of hyperglycemia on ADM and its receptors in β-cells, we treated NIT-1 cells with increasing doses of glucose for 24 hours. As shown in Fig 4, increases in glucose concentrations from 5.6 mM to 22.2 mM resulted in a dose-dependent stimulation in mRNA expressions for CRLR, RAMP2 and RAMP3 (P<0.05), whereas the mRNA expressions for ADM were not significantly affected by the glucose treatment. These results indicated that increased glucose levels in GDM may contribute, at least in part, to the enhanced pancreatic βcell ADM receptor expressions, thus enhancing ADM actions.

ADM inhibits NIT-1 β-cell proliferation, and this inhibition is blocked by ADM antagonist
To assess the influence of ADM on β cell growth, we treated NIT-1 cells in Ham's F12K medium containing 16.7 mM glucose with ADM (10 −9 M) in the presence or absence of ADM22-52 (10 −8 M) for up to 5 days (Fig 5). The micrographs showed a clear reduction in cell density after 5 days of ADM treatment, and this decrease was reversed by pretreatment of the cells with ADM22-52. Furthermore, the MTT reduction test demonstrated that ADM did not affect the cell proliferation in medium containing 5.6 mM glucose (Fig 6), but did inhibit cell proliferation in medium containing 16.7 mM glucose in a dose-dependent manner, and this inhibition was reversed by ADM22-52, confirming that the inhibitory action of ADM on NIT-1 cell proliferation is mediated through ADM receptors.

ADM inhibits glucose-induced insulin mRNA, synthesis and secretion by NIT-1 β-cells
To determine the effects of ADM on insulin mRNA expressions by NIT-1 cells, we treated the cells with increasing doses of glucose in the presence or absence of ADM (10 −9 M) for 24 hours (Fig 7A). The results showed that glucose stimulates NIT-1 cell insulin mRNA expression in a dose-dependent manner, whereas ADM inhibits this expression starting from 11.1 mM of glucose incubation. In addition, raising glucose from 5.6 to 16.7 mM led to an approximately 150% increase in insulin concentration after 30-min glucose stimulation (Fig 7B), and the addition of ADM to NIT-1 cells resulted in a dose-dependent reduction of insulin secretion. This inhibition reached 75% for an ADM concentration of 10 -8 M. In addition, to determine the underlying mechanisms of ADM actions, NIT-1 cells were pretreated with ADM antagonist ADM22-52, adenylyl cyclase inhibitor SQ22536, and Erk pathway inhibitor P98059 prior to the addition of ADM (Fig 7C). The results showed that ADM dose-dependently inhibits insulin synthesis in the cell culture medium after 24 hours treatments. The inhibitory action of ADM on insulin synthesis was reduced by ADM22-52, SQ22536, and PD98059, implying that ADM receptors, adenylyl cyclase, and Erk pathway are involved in the ADM inhibitory actions.

ADM upregulates ER stress biomarker genes
Next, we assess the impact of raising glucose and ADM treatment on ER stress in NIT-1 β cells. NIT-1 cells were incubated in culture medium containing 5.6 to 22.2 mM of glucose in the presence or absence of ADM (10 −9 M) for 24 hours, and ER stress markers were determined by using Real-time PCR. As shown in Fig 8, incubation of NIT-1 cells with increasing dose of glucose does not significantly affect the expression of t-Xbp-1, s-Xbp-1, Chop, and BIP, suggesting that these ER stress makers were not stimulated and Xbp-1 splicing was not activated by increasing glucose treatment. In contrast, addition of the ADM (10 −9 M) in the culture medium led to an approximately 9-fold increase in mRNA for t-Xbp-1 compared to glucose-matched controls (P<0.01). Further, ADM significantly stimulated BIP mRNA expression, and this stimulation reached up to 10-fold higher than glucose-matched controls (P<0.01). These results suggest that ADM but not raising glucose causes activation of ER stress, and that is not accompanied by Xbp-1 splicing.

Discussion
GDM is characterized by reduced adaptation of the pancreatic β-cells to the increased demands during pregnancy [24], but the molecular basis for these alterations remains unclear. The present study utilized pancreatic tissues from non-pregnant and pregnant mice, insulin producing β-cell line, and isolated mouse pancreatic islets to demonstrate that: 1) ADM and CRLR, RAMP2 and RAMP3 are abundantly expressed in mouse pancreatic islets and co-localized with insulin. 2) ADM block glucose stimulated insulin secretion in mouse pancreatic islets.
3) The expressions of ADM, CRLR and RAMP2 in islets from pregnant mice are lower compared to that of non-pregnant mice. 4) NIT-1 β-cells express mRNA for ADM and CRLR, RAMP 2, and RAMP3, and the expressions for receptor components, but not ADM, are stimulated by glucose in a dose-dependent manner. 5) ADM inhibits NIT-1 cell proliferation, and this inhibition is reversed by ADM antagonist, ADM22-52. 6) ADM inhibits NIT-1 cells glucose-induced insulin synthesis and secretion 7) these inhibitory effects were reversed by ADM22-52, adenylyl cyclase inhibitor, SQ22536, and Erk pathway inhibitor PD98059 in NIT-1 cells, and 78) Treatment of NIT-1 cells with ADM resulted in upregulation of ER stress biomarker genes, BIP and t-Xbp-1, indicating the involvement of ER stress in ADM's action. We therefore propose that ADM maybe involved in the regulation of β-cell functions in an

PLOS ONE
Adrenomedullin suppresses β-cell insulin secretion  autocrine and paracrine manner. Reduced ADM and its receptors expression during pregnancy may play a role in promoting β-cell mass and insulin production, while increased plasma ADM in GDM may contribute to the β-cells dysfunction, and blockade of ADM may reverse β-cell insulin production.

The cellular localization of ADM system in pregnant mouse pancreas
Four major cell types, α, β, δ and PP cells are found in the endocrine pancreas, and secrete hormones into the bloodstream [25]. The α cells produce glucagon and β cells produce insulin, to maintain glucose homeostasis. The δ cells produce somatostatin and PP cells produce pancreatic polypeptide that modulate the secretory function of the other cell types. It has been reported that ADM appears at day 11.5 in rat development coinciding in pancreas with glucagon and insulin in the same cells [26]. While at some time in rat development, all the cell type express ADM and then progressively evolve towards the adult pattern, where only the PP cells display a strong immunoreactivity for ADM. Meanwhile, the mRNA for ADM and its receptors have been shown to exist in six different insulin-producing cell lines, including RINm, N289, TR4, CRL2057, CRL1777, and CRL2055 [17], suggesting that the expression of ADM and its receptors in endocrine pancreatic cells seems to be a highly conserved feature from both the phylogenetic [27] and the ontogenetic [26] perspective. In the present study, we demonstrated that ADM and its receptor components CRLR, RAMP2, and RAMP3 are expressed in the pancreatic islets of non-pregnant and pregnant mice and co-localized with insulin (Figs  1 and 2). This finding was confirmed by immunofluorescence in the pancreatic tissues and by molecular analysis of mRNA expression in an insulin-producing mouse NIT-1 β-cell line. The pattern of distribution and endocrine cell type in the mouse are in an agreement with those previously reported by other groups [17,28]. The co-localization of ADM and its receptors in the pancreas implicates that ADM may play a role in the control of both normal and altered pancreatic physiologies. Further, the expressions for ADM, CRLR and RAMP2, but not RAMP3, are significant reduced in islets from pregnant mice when compared to that of nonpregnant mice, this may imply that alteration of ADM system in mouse pancreas may play a role in pregnancy-related β-cell adaptation. In addition, our results in mouse NIT-1 cell line showed that glucose dose-dependently increased mRNA expressions of CRLR, RAMP2 and RAMP3, but not ADM per se (Fig 4). These findings provide new insight into the association between hyperglycemia and enhanced ADM action in diabetic pregnancies, indicating that glucose intolerance in GDM may be one of the stimulants for the enhanced ADM influence through increased ADM receptor on β-cells, thus contributing to the impaired insulin secretion.

ADM inhibits β-Cell growth and glucose-stimulated insulin secretion
Conflicting results have been reported regarding the effect of ADM on insulin secretion. ADM has been reported to stimulate [29] or inhibit [17] insulin secretion in isolated rat islets. We report here that ADM inhibited glucose stimulated insulin secretion in pancreatic islets isolated from pregnant mice, further confirming previous findings in rat [17] and corroborating our previously published work using a human β-cell line [15]. However, isolated pancreatic islets used in these studies consist of various cell types and thus may not be appropriate in evaluating the impacts of ADM on β-cell functions as well as ADM mechanisms of action. Therefore, in this present study, we utilized a differentiated mouse insulinoma cell line NIT-1 to determine the direct effect of ADM on β-cell growth and insulin secretion and synthesis and investigate the mechanisms of ADM action on β-cell growth and function. This NIT-1 cell line retains various differentiated features of native β-cells [30] and is considered a suitable model for studying the β-cell proliferation, insulin secretion and biosynthesis [31]. Here, we show that ADM did not affect the cell proliferation in medium with 5.6 mM glucose, but did inhibit cell proliferation in medium with 16.7mM glucose in a dose-dependent manner (Figs 4 and 5), and this inhibition was blocked by pretreatment of cells with ADM22-52, indicating that enhanced ADM inhibitory action in hyperglycemia may result from glucose-stimulated ADM receptor overexpression, and the ADM inhibitory effect are mediated through ADM receptors. In addition, our results showed that glucose stimulates NIT-1 cell insulin mRNA expression as well as insulin secretion in a dose-dependent manner (Fig 6A and 6B), and ADM inhibits both glucose-stimulated insulin mRNA expression and secretion. Furthermore, ADM dose-dependently inhibits insulin synthesis in the cell culture medium after 24 hours treatments (Fig 6C).
Our results are consistent with the effects of ADM on β-cells described by other groups in rat models. Martinez et al reported that a monoclonal antibody against ADM increases insulin release 5-fold more than controls in isolated rat islets [17]. Sekine et al showed that pancreatic beta cells ADM exposure resulted in a reduction in insulin secretion [32]. ADM administration in diabetic SHR/ N-cp rats in vivo decreases serum insulin levels with a concomitant increase in circulating glucose levels [33]. Such experimental evidence may implicate ADM as a fundamental factor in maintaining insulin homeostasis and normoglycemia, and dysregulation of ADM maybe one of the causal factors in diabetes. Collectively, further investigation focused on the development of blocking agents for ADM may result in new treatments for pancreatic ADM-related disorders.

Underlying mechanisms of ADM actions
Glucose stimulates insulin secretion by generating metabolic coupling factors which promote β-cell exocytosis of insulin granules [34]. In physiological circumstances, mitochondrial metabolism of glucose increases ATP levels and leads to closure of the ATP-sensitive K+ channel, thereby resulting in the β-cell membrane depolarization. The opening of the voltagegated calcium currents (VGCC) then induces Ca2+ influx into the β-cell and promoting insulin exocytosis. The mechanisms of ADM inhibitory actions on β-cell functions may include the regulation of glucose metabolism that is related to the β-cell excitability, the membrane potential or the VGCC, intracellular cAMP concentration, and the exocytosis machinery of insulin granules. In the present study, we observed that the inhibitory action of ADM on insulin synthesis was abolished by ADM22-52, SQ22536, and PD98059 ( Fig 6C). The effects of ADM22-52 suggest that the effects seen were due to ADM-ADM receptor interactions and not a non-specific toxic effect of ADM on β-cells. The finding that SQ22536 reverses ADM inhibited insulin release supports the notion that cAMP is the second messenger for the ADM actions [7], and confirm that exocytosis of insulin granules is stimulated through the elevation of cAMP [35]. PD98059 blocks ADM actions, indicating that the members of the mitogenactivated protein kinase family, extracellular response kinase-1/2 (Erk1/2), are activated in the β-cell after ADM addition [36]. Therefore, a better understanding of the ADM system in pancreatic physiology and pathological states, such as GDM, may help define new areas of therapeutic intervention to improve the metabolic homeostasis.

ADM induces ER stress in β-cells
Physiologically required for the folding, export, and processing of newly synthesized insulin, pancreatic beta cells possess a highly developed ER [37]. Served as an ER chaperone and a sensor of protein misfolding, immunoglobulin heavy chain binding protein (BIP) plays a central role in this process [38]. ER stress developed and apoptosis is induced by enhanced transcription of C/EBP homologous protein (CHOP) [39,40], and X-box binding protein 1 (Xbp-1) is activated when misfolded proteins are accumulated in the ER [41]. Increasing evidence indicates that ER stress may be directly involved in the β-cell dysfunction and death observed during the development and progression of diabetes mellitus [42], and overwhelming ER stress can also induce oxidative stress [43], which may further impair β-cell function. In the present study, our Real time-PCR analysis showed significant upregulation of ER stress genes t-Xbp-1 and BIP (Fig 7), suggesting that ADM is capable of activating ER stress and dysregulating the unfolded protein response (UPR) pathway, which inevitably leads to marked β-cell dysfunction. We also showed decreased β-cell growth and proliferation after exposure to ADM (Figs 4 and 5), further confirming ER stress-mediated apoptosis. Therefore, we propose from these data that ADM induces excessive activation of ER stress and subsequent failure of the UPR, may contribute to eventual β-cell dysfunction.
Further studies are required to examine if administration of ADM to pregnant mice could induce GDM symptoms with impaired β-cell functions, whether reduced number of β-cells in pancreas islets and lowered circulating insulin concentration in non-obese GDM mice are associated with increased ADM and its receptor expression in pancreatic islets, and if administration of ADM antagonist could mitigate impaired insulin production and glucose intolerance in vivo. Nonetheless, our current study provides the evidence that ADM could be involved in both the physiological regulation of insulin secretion in normal pregnancy and the pathogenesis of diabetic pregnancy as a causative factor of impaired insulin synthesis and secretion, and blockade of ADM actions in GDM patients with its antagonists may improve βcell functions.